Herpes simplex virus (HSV) is a very common human pathogen that infects epithelia and skin before spreading to sensory neurons where it establishes a lifelong infection. Entry of HSV is a complex molecular process. One essential step is the interaction of HSV glycoprotein D (gD) with a cellular receptor to induce fusion of the viral envelope with the plasma membrane or an endosomal membrane. One of the main gD-receptor on neurons and epithelial cells is the cell adhesion molecule nectin-1. The long-term objectives of this study are to explain how the virus exploits cellular pathways to reach nectin-1 at cell junctions, stimulate endocytosis and fuse with the host membrane. Two specific aims are proposed to achieve these objectives. In aim 1 the cellular pathways that are activated when gD binds to nectin-1 during HSV entry will be defined. Nectin-1 expressing cells will be exposed to either membrane bound or soluble forms of gD to identify cellular responses involved in receptor internalization. The signals elicited by gD binding to nectin-1 will be compared to those of a natural ligand, nectin-3, which interacts with nectin-1 at cell junctions and at synapses. Finally, pathways involved in receptor endocytosis will be correlated with pathways activated during HSV entry. In aim 2 the effect of HSV entry on the integrity of cellular junctions will be defined. Since gD binding affects nectin-1-mediated cell adhesion, the effects of gD on the architecture of cell junctions will be studied. The viral and cellular needs for HSV particles to surf on the plasma membrane and access nectin-1 at cellular junctions will be defined. The importance of this movement for entry into polarized epithelial cells will be analyzed. This project will extend studies of HSV attachment and binding to receptors toward novel aspects of the immediate cellular responses induced by the virus. State of the art technology and new sets of reagents will be used to explore the cellular mechanisms triggered by HSV binding to the cell surface. Innovative approaches developed in this project will allow studies of HSV entry at cellular junctions by direct observation of viruses and cell components in real time using live cell confocal microscopy. This study will increase our understanding of the early steps of HSV infection and identify new targets for antiviral therapeutic actions. Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect 80% and 20% of all American adults respectively and current treatments do not reduce the occurrence of HSV reactivation and transmission, thus new therapeutic strategies need to be developed. This study will determine the immediate consequences of the virus binding to the cell and identify cellular mechanisms involved in virus entry. By identifying new requirements for virus entry in the host, this study will provide targets for novel therapeutic approaches to interfere with this process. [unreadable] [unreadable] [unreadable]